1. Absence of CCR4 Exacerbates Skin Inflammation in an Oxazolone-Induced Contact Hypersensitivity Model 
Sari Lehtimäki, Sari Tillander, Anne Puustinen, Sampsa Matikainen, Tuula Nyman, Nanna Fyhrquist, Terhi Savinko, Marja-Leena Majuri, Henrik Wolff, Harri Alenius, Antti Lauerma 
Journal of Investigative Dermatology 
Volume 130, Issue 12, Pages (December 2010)
DOI: /jid
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Ear tissue from vehicle (VEH) and oxazolone (OXA)-treated mice 24hours after OXA elicitation. (a) Ear swelling is significantly increased in OXA-treated CCR4-/- mice compared with wild-type (WT). (b) Hematoxylin–eosin staining of ear tissue from OXA-treated WT and CCR4-/- mice reveals increased edema and inflammatory cell infiltration in CCR4-/- mice compared with WT mice. Bar=100μm. (c) Number of total inflammatory cells, lymphocytes, eosinophils, and neutrophils in the ear tissue of VEH-treated and OXA-treated mice. *P<0.05, **P<0.01, ***P<0.001; bars represent mean+SEM, n=8. HPF, high power field.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
CD4+ and CD8+ cells in the skin. (a) According to immunohistochemical stainings, increased numbers of CD4+ cells have infiltrated the skin of oxazolone-sensitized and challenged CCR4-/- mice compared with wild-type (WT) mice, although there are no differences in the number of CD8+ cells. Ratio of CD4+/CD8+ cells is increased in CCR4-/- mice compared with WT as evidenced by (b) immunohistochemistry (IHC) stainings and (c) flow cytometric (FC) analysis. *P<0.05; bars represent mean+SEM, n=4–6. HPF, high power field.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Cytokine expression in the skin. (a) Real-time PCR analysis of ear tissue 24hours after elicitation with oxazolone. Significantly elevated mRNA expression of proinflammatory cytokines IL-1β, IL-6, and tumor necrosis factor (TNF)-α in oxazolone (OXA)-treated groups versus vehicle-treated mice. In addition, IL-6 and TNF-α are significantly more expressed in the CCR4-/- OXA group compared with the WT (wild-type) OXA group. (b) The mRNA expression of Th1 (IL-12p35) and Th2 (IL-4, IL-13) cytokines shows a similar pattern to proinflammatory cytokines, n=8. (c) Similar percentages of CD3+ cells express IL-13 and IFN-γ in phorbol myristate acetate and ionomycin-stimulated skin cells from OXA-sensitized and challenged WT and CCR4-/- mice, n=3. *P<0.05, **P<0.01, ***P<0.001; bars represent mean+SEM. RU, relative units.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
The mRNA expression analysis of ear tissue. (a) P- and E-selectins, (b) multiple chemokines (CCL3, CCL4, CCL5, CCL8, CCL17, and CCL22), and (c) corresponding chemokine receptors (CCR3, CCR4, and CCR5) 24hours after oxazolone (OXA) elicitation. *P<0.05, **P<0.01, ***P<0.001; bars represent mean+SEM, n=8. RU, relative units; VEH, vehicle; WT, wild type.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
CCL27 and CCR10 expression in the skin and chemotaxis of draining LN cells. (a) The mRNA expression analysis of skin samples shows no differences in the expression levels of CCL27 or CCR10, n=8. (b) Flow cytometric analysis of CD3+ cells in the skin show that the same proportion of T cells is positive for CCR10 expression in the oxazolone (OXA)-sensitized and challenged wild-type (WT) and CCR4-/- mice. Gated on CD3+ cells. (c) CCR4-/- mice show severely impaired migration toward CCL17 and CCL22, whereas the chemotactic response toward CCL27 is similar to that of the WT. Bars represent mean+SEM. RU, relative units; VEH, vehicle.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

7. Figure 6
The number of CD3+CD4+ and CD3+CD8+ cells in the draining LNs of wild-type (WT) and CCR4-/- mice. (a) 0, 2, 4, and 7 days after sensitization (n=2–3) and (b) 0, 4, 12, and 24hours after elicitation (n=2–3). (c) Ear swelling response 2, 4, 6, 12 (n=2–3), and 24hours after elicitation (n=8) *P<0.05, **P<0.01; bars represent mean+SEM.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

8. Figure 7
IL-13 and IFN-γ expression in the stimulated draining LN cells. (a) The number of CD3+IL-13+ and CD3+IFN-γ+ cells and (b) mRNA expression of IL-13 and IFN-γ in the stimulated lymph node cells of oxazolone-sensitized and challenged wild-type (WT) and CCR4-/- mice at 0, 4, 12, and 24hours post-elicitation, n=2–3. RU, relative units.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

9. Figure 8
Foxp3 expression in the skin and draining LN cells. (a) According to mRNA analysis from the skin at 24hours post-challenge, Foxp3 and TGF-β are significantly upregulated in the CCR4-/- mice, whereas there are no differences in the IL-10 expression, n=8. (b) Flow cytometric (FC) and immunohistochemical (IHC) analysis reveal an increased number of Foxp3+ cells in the skin of oxazolone-sensitized and challenged CCR4-/- mice compared with wild-type (WT), n=3 (FC), n=4 (IHC). (c) The number of CD3+Fox3+ cells in the draining lymph nodes (LNs) follows the kinetics of CD3+CD4+ cells, in both the WT and the CCR4-/- mice, n=2–3. (c) *P<0.05, **P<0.01, ***P<0.001; bars represent mean+SEM. RU, relative units.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions